Rickettsia rickettsii is the causative agent of one of the most severe tick-borne diseases in the United States. The compounding factors of rickettsial pathogenicity and route of tick transmission (horizontal and vertical) influence the ecology and epidemiology of human rickettsioses. Our long term goal is to elucidate the molecular mechanisms that are central to rickettsial transmission by ticks. Despite the overlapping distribution of ticks with multiple Rickettsia species in nature, individual tick species are not infected with a diverse range of rickettsial species. Therefore, the broad hypothesis being tested is that unique tick-derived factors are required for successful horizontal and vertical transmission of spotted fever group (SFG) Rickettsia. Preliminary evidence indicates rickettsial transmission is dependent on the appropriate tick/Rickettsia relationship and the pathogenic nature of the Rickettsia. It is also known that ticks actively respond to the infecting rickettsiae;however, the extent to which modulation of tick-derived molecules is required for rickettsial survival within the tick is not known. Thus, the experimental focus of this proposal is to delineate the specificity of tick/Rickettsia relationships, the relationship between rickettsial pathogenicity and transmission, and to define the tick-derived molecules associated with rickettsial infection via comparative analysis of different tick and Rickettsia species. The previously undefined parameters of tick/Rickettsia interactions will be determined in the following specific aims. 1) Characterize the horizontal and vertical transmission of pathogenic and nonpathogenic SFG Rickettsia in tick hosts in vivo. The previously unknown kinetics of transmission for six species of SFG Rickettsia will be assessed in Amblyomma and Dermacentor ticks. Horizontal transmission of rickettsiae from Rickettsia-infected ticks to vertebrate hosts will be determined using a laboratory model of tick infestation. The efficiency of vertical transmission of Rickettsia will be assessed by fluorescence in situ hybridization and quantitative real-time PCR (qPCR) and the influence of SFG Rickettsia carriage on tick fitness will be assessed. 2) Examine the interaction between tick-derived molecules and SFG Rickettsia during infection in typical and atypical tick/Rickettsia relationships. Protein-protein interaction assays will be used to identify rickettsial binding proteins present in whole tick tissues. Studies will utilize paired tick tissues and SFG rickettsiae in comparative pull-down and protein binding assays. We will identify key tick proteins and delineate their role in the establishment of Rickettsia via bioassays including adhesion/invasion blocking assays, immune/antimicrobial activity assessment, and RNA-mediated interference (RNAi). The utilization of specific molecules by individual species of Rickettsia will also be assessed. PUBLIC HEALTH RELEVANCE Despite the overlapping distribution of ticks with multiple Rickettsia species, in nature, individual tick species are not infected with a diverse range of rickettsial species. We will examine the specificity of tick/Rickettsia relationships and delineate the molecular mechanisms of tick vector competence for spotted fever Rickettsia to better understand the ecology and epidemiology of tick-borne rickettsial diseases.